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BRCA2
Mutation Research 601 (2006) 191–201
Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2 Barbara C. Godthelp a , Paul P.W. van Buul a , Nicolaas G.J. Jaspers b , Elhaam Elghalbzouri-Maghrani a , Annemarie van Duijn-Goedhart a , Fr´e Arwert c , Hans Joenje c , Małgorzata Z. Zdzienicka a,d,∗ a
Department of Toxicogenetics, Leiden University Medical Center, Building 2, Postzone S-6-P, P.O. Box 9600, 2300 RC, Leiden, The Netherlands b Department of Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands c Department of Clinical Genetics and Human Genetics, Free University Medical Center, Amsterdam, The Netherlands d Department of Molecular Cell Genetics, Collegium Medicum, N.Copernicus University, Bydgoszcz, Poland Received 31 January 2006; received in revised form 4 July 2006; accepted 11 July 2006 Available online 21 August 2006
Abstract Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway. © 2006 Elsevier B.V. All rights reserved. Keywords: Fanconi anemia; BRCA2; Radioresistant DNA synthesis; DNA damaging agents; Chromosomal instability
1. Introduction FA is an autosomal recessively and X-linked inherited disorder characterized by multiple congenital abnormalities, abnormal skin pigmentation, progressive
∗
Corresponding author. Tel.: +31 71 5269614; fax: +31 71 5268284. E-mail address: [email protected] (M.Z. Zdzienicka).
0027-5107/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2006.07.003
bone marrow failure and a marked predisposition to develop cancer, primarily acute myeloid leukemia and squamous cell carcinomas of the head and neck. The cellular hallmark of FA is genomic instability and a specific hypersensitivity to DNA cross-linking agents such as MMC, which is used as a clinical diagnostic test for FA [1]. At least twelve different complementation groups can be discerned on the basis of complementation studies [2,3]. For 10 of these groups
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the disease-causing genes have been identified: FANCA, -B, -C, -D1/BRCA2, -D2, -E, -F, -G, -J and -L [2,4–8]. Recently, another FA gene was identified, FANCM, on basis of protein complex purification [9]. Despite the cloning of these FA genes, the precise function of the FA pathway is still largely unknown. The FA proteins are thought to function in a DNA damage response pathway involving the breast cancer susceptibility gene products, BRCA1 and BRCA2 [7,10]. A key step in this pathway is the activation of FANCD2 by monoubiquitination through FANCL [5] for which the nuclear multiprotein core complex consisting of FANCA, -B, -C, -E, -F, -G, -L and -M is required [2,5,6,9,11]. After monoubiquitination, FANCD2 is redistributed to nuclear foci containing BRCA1 and Rad51, linking FA with HR repair [10]. Moreover, the direct interaction of FANCA with BRCA1, of FANCG with FANCD1/BRCA2 and the interaction between ATM and FANCD2 [12–14], are also reminiscent of a role for the FA pathway in DNA repair, either directly or indirectly. Evidence for a more structural role of FA proteins comes from a model in which the function of FANCD2 monoubiquitination is to stabilize structurally blocked and broken replication forks prior to repair by specialized DNA damage repair pathways [15]. Since FA-D1/BRCA2 and FA-J patient derived cells display normal FANCD2 monoubiquitination following DNA damage, it is likely that these genes function independent of the FA core complex-FANCD2 pathway [3,7]. The recently identified genes, FANCJ and FANCM, both contain potential enzymatic domains belonging to the family of DEAH helicases or DNA stimulated ATPases and they are therefore likely to unwind and/or directly interact with DNA [8,9]. Germline mutations of the breast cancer susceptibility gene BRCA2 are associated with a predisposition to breast cancer but other cancers including ovarianand pancreatic-cancer are also seen in BRCA2 mutation carriers [16–18]. The BRCA2 protein is important in cellular responses to DNA damage and in maintaining genomic integrity, while a role in cell cycle progression has been suggested [19,20]. Crystal structure analysis revealed that BRCA2 is involved in Rad51-mediated HR as a stimulator that helps to displace RPA and facilitates ssDNA–Rad51 nucleoprotein filament formation [21,22]. Moreover, BRCA2 functions as a molecular switch since a decrease in BRCA2 phosphorylation stimulates its interaction with Rad51 [23]. Studying the role of BRCA2 in vivo has been proven difficult since knockout mice are embryonic lethals [24] but viable mice that harbor hypomorphic Brca2 mutations or conditional knockouts have been generated [25-27]. Until recently, CAPAN-1 derived from a pancreatic epithelial tumor
was the only well characterized human cell line defective in BRCA2 [28,29]. However, with the discovery that FAD1 patients carry biallelic mutations in BRCA2 [7], the first primary human BRCA2-deficient lines have become available, two of which are characterized in the present study (Table 5). These FA-D1/BRCA2 fibroblasts were found to be severely impaired in the formation of nuclear foci that contain the central HR protein, Rad51, a defect that has not been observed in any other FA complementation group [30–32]. Rodent and chicken cells that are deficient in HR genes, including BRCA2, display a severe genome instability phenotype with impaired DNA damage induced Rad51 foci formation, an excessive sensitivity to various DNA damaging agents, an increased frequency of spontaneous and MMC-induced chromatid-type chromosomal aberrations (CA) and a reduced capacity to form sister chromatid exchanges (SCE) [33–38]. To determine whether the phenotype of FAD1/BRCA2 cells conforms with that of HR repairdeficient cells or with that of “typical” FA cells, we analyzed the cellular characteristics of human BRCA2deficient fibroblasts and B-cells derived from two unrelated patients. As these cells displayed features that are considered characteristics of both FA- and HR-deficient cells, our results suggest that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls a separate pathway that is more directly linked to HR-based repair. 2. Materials and methods 2.1. Cell culture Primary fibroblasts were cultured in plastic dishes (P94 ; Greiner) in Ham’s F10 medium (GIBCO) without hypoxanthine and thymidine or in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (GIBCO), supplemented with 10% fetal calf serum (Bodinco), penicillin (100 U/ml) and streptomycin (0.1 mg/ml). Cells were maintained at 37 ◦ C in a 5% CO2 atmosphere, humidified to 95–100%. For subcultures the cells were detached by use of 0.25% trypsin containing 0.02% EDTA. The primary fibroblast strains used were: wild type (FN1, VH10), FA-A (EUFA432), FA-C (EUFA449), FA-D1 (EUFA423, F145), FA-D2 (EUFA8, EUFA1284, EUFA1289), FA-E (EUFA622), FA-F (EUFA121), AT (AT5BIVA) and the SV40-immortalized fibroblasts: wild type (MRC5i, VH10i), FA-D2 (PD733i) and FA-D1 (EUFA423i). B lymphoblastoid cell lines (BLCL): wild type (JVM, TK6, RAJI), FA-A (HSC72) and FA-D1 (HSC62, EUFA423-L) were grown in 75 cm2 culture flasks (Greiner) in RPMI 1640 medium Dutch modification (GIBCO) supplemented with GLUTAMAX I (GIBCO), 20 mM sodium pyruvate (GIBCO), 10% fetal calf serum (Bodinco), penicillin (100 U/ml) and streptomycin
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(0.1 mg/ml). BLCLs were maintained at 37 ◦ C in a 5% CO2 atmosphere, humidified to 95–100%. 2.2. Chemicals MMS was obtained from Merck, Darmstad, Germany; MMC from Kyowa, Hakko Kogyo, Tokyo; bleomycin from Dagra Pharma B.V.; polyethylene glycol (PEG, 1450 MW) and 5-bromo-2 -deoxyuridine (BrdUrd) were purchased from Sigma Chemical Co.
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sion was added to a monolayer of recipient cells (1.0 × 106 mutant cells per P94 dish) and allowed to attach at room temperature for 15 min. The cells were fused by treatment with 1 ml of 47% PEG in serum-free medium containing 10% DMSO for 1 min, followed by washing in serum-free medium containing 5% DMSO. After 24 h the cells were trypsinized and split to five P94 dishes with selective medium containing G418 (Gibco BRL). The resulting microcell hybrid clones were isolated after 14–28 days. 2.7. Analysis of CA, SCE
2.3. Irradiation For X-ray irradiation cells were irradiated in tissue culture medium at a dose rate of 2.98 Gy/min (200 kV, 4 mA, 1 mm Al). For gamma irradiation cells were irradiated with a 137 Cs source in culture medium at a dose rate of 0.8 Gy/min. For UVC irradiation (254 nm), a Philips TUV germicidal lamp was used with a fluence rate of 0.19 W/m2 , measured with the IL/770 germicidal radiometer; cells were irradiated in PBS. 2.4. Clonogenic survival assays Cultures in exponential growth were trypsinized and 500– 2000 fibroblasts were plated in P94 dishes in duplicate (controls in triplicate) left to attach for 4 h, and then irradiated or exposed to MMS for 1 h, bleomycin for 24 h and continuously to MMC, in complete medium. After treatment with chemicals the cells were washed twice with PBS, with the exception of MMC, and returned to fresh medium. After 14–17 days the dishes were rinsed with 0.9% NaCl, dried, stained with methylene blue (0.25%) and colonies were counted under a light microscope (LW Scientific). In all experiments, wild type fibroblasts were treated in an identical manner to serve as controls. 2.5. Measurement of radioresistant DNA synthesis (RDS) after ␥-irradiation Measurement of RDS was performed essentially as described but without the trichloroacetic acid precipitation step [39]. In short, cells were seeded in duplicate 30mm dishes (four for the unirradiated control) and were pre-labeled overnight with [14 C]-thymidine (Amersham) in HEPES-buffered thymidine-free Ham’s F10 medium and then exposed to graded doses of 137 Cs ␥-irradiation (1.2 Gy/min) and subsequently labeled with [3 H]-thymidine (Amersham) for 4 h. Free thymidine pools were chased by a further 30–45 min incubation in unlabeled medium. Scintillation counted 3 H/14 C radioactivity ratios of alkali-lysed cells were taken as a measure of DNA synthesis rates and plotted as percentages of unirradiated cells. 2.6. Microcell-mediated chromosome transfer Microcells with a single human chromosome 13 were obtained as described previously [38]. The microcell suspen-
Exponentially growing wild type and FA patient derived BLCL were either mock-treated or treated for 2 h (SCE) with MMC or for 24 h (CA) with 2, 3.3, 4, 5, 10, 16.5, 40, 80, 320 and 640 ng MMC/ml (wt) or with 1.0, 1.65, 2.0 and 3.3, 4, 10, 20 and 40 ng MMC/ml (FA cells). For SCE analysis, BrdUrd was added to the medium (10 M final concentration) to enable sister chromatid differentiation. The cells were harvested for CA analysis 24 h and for SCE analysis 51–72 h after (mock-) treatment, including 2 h incubation with colcemid (1 g/ml final concentration). The cells were fixed in ethanol:glacial acetic acid (4:1) after treatment with hypotonic solution (0.05 M KCl). Air-dried preparations were made and stained with a 5% aqueous Giemsa solution for 5 min to analyze CA and with FPG to visualize SCE [40]. For CA 100 mitotic cells were scored at each dose, while SCE were counted in 25 cells. Data for CA and SCE analyses are from at least two independent experiments of which the mean and the SEM were calculated. Statistical analysis was performed using t-tests.
3. Results 3.1. FA-D1/BRCA2 fibroblasts: population doubling times and cloning efficiencies We determined the cloning efficiency (CE) and population doubling time of FA-D1/BRCA2 primary fibroblasts to establish whether these FA-D1/BRCA2 cells resemble fibroblasts from other FA complementation groups. The data revealed that FA-D1/BRCA2 fibroblasts had a relatively low CE, i.e., 4.4 ± 0.9% (EUFA423) and 2.9 ± 0.98% (F145), respectively (Table 1), whereas all other FA fibroblasts showed CEs ranging from 8% in EUFA8 (FA-D2) to 26.7% in EUFA121 (FA-F), which is in range of wild type primary fibroblasts. In FA-D1 SV40-immortalized fibroblasts (EUFA423i) we also observed a lower CE that could be complemented to wild type level by transfer of a single human chromosome 13 providing the BRCA2 gene (Table 1). These FA-D1 cells with an additional chromosome 13 showed full complementation for MMC-sensitivity in cell survival and DNA damage induced nuclear Rad51 foci formation (results not shown). In addition, we
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Table 1 Low cloning efficiency in FA-D1/BRCA2 primary and immortal fibroblasts Cell line
Complementation group
Cloning efficiency (CE)a ±S.E.M.
VH10 VH25 FN1
wt wt wt
9.4 ± 1.7 18.9 ± 3.3 20.3 ± 3.4
MRC5ib VH10i
wti wti
38.5 ± 2.4 45.2 ± 17.6
EUFA432
FA-A
15.9 ± 2.1
EUFA449
FA-C
18.4 ± 2.5
EUFA423 F145
FA-D1/BRCA2 FA-D1/BRCA2
EUFA423i EUFA423i/#13
FA-D1/BRCA2i FA-D1 complemented
21.1 ± 1.6 34.7 ± 4.6
EUFA8 EUFA1284 EUFA1289
FA-D2 FA-D2 FA-D2
8.0 ± 0.53 22.0 ± 2.0 20.9 ± 1.8
PD733i
FA-D2i
34.0 ± 2.2
EUFA622
FA-E
23.5 ± 6.5
EUFA121
FA-F
26.7 ± 4.3
4.4 ± 0.90 2.9 ± 0.98
a The cloning deficiency was determined from at least six experiments. Results are the means ± S.E.M. b i, SV40-immortalized fibroblasts.
found that FA-D1/BRCA2 primary fibroblasts as well as FA-D2 fibroblasts have approximately a 2.3-fold longer population doubling time (≈135 h) than wild type fibroblasts (≈55 h), whereas e.g., FA-A fibroblasts exhibited an approximately 1.2-fold longer population doubling time (≈67 h). These results may suggest that primary cells carrying biallelic hypomorphic mutations in BRCA2 (Table 5) are more severely affected than cells that carry null mutations in any other FA gene. 3.2. Sensitivity to DNA damaging agents To determine cross-sensitivity of the FA-D1/BRCA2 fibroblasts to various DNA damaging agents, we compared the clonogenic survival of these cells in response to these agents with that of fibroblasts from other FA groups. FA-D1/BRCA2 as well as FA-D2 primary fibroblasts were approximately 5- (when compared to FN1) to 17-fold (when compared to VH10) more sensitive to MMC than wild type fibroblasts (Fig. 1), based on the dose required to reduce the survival to 10% (D10 ). Furthermore, we found no significant sensitivity to X-ray irradiation in fibroblasts from the FAD1/BRCA2 and FA-D2 groups (Fig. 1) when compared
with fibroblasts from an ataxia telangiectasia patient (AT5BIVA), which were ∼3-fold more sensitive than wild type fibroblasts. Similar observations were made for FA-A, FA-C and FA-J primary fibroblasts (results not shown). The only exception was EUFA8 (FA-D2) fibroblasts that showed a mildly increased sensitivity to X-ray irradiation (1.6-fold), which indicates that X-ray sensitivity in clonogenic survival is not a general feature of FA fibroblasts. Surprisingly, both FA-D1 and FA-D2 fibroblasts displayed a slightly increased sensitivity to the radiomimetic agent bleomycin (2-fold), which did not correspond with the lack of an increased X-ray sensitivity in clonogenic survival in these cells (Fig. 1). The FA-D1 and FA-D2 fibroblasts both showed a slightly increased sensitivity to the monofunctional alkylating agent MMS (∼1.4-fold), whereas only FA-D1 fibroblasts showed a marked sensitivity to UV irradiation (∼2-fold). Fibroblasts derived from FA-A, FA-E and FA-J complementation groups were also slightly sensitive to MMS (∼1.2–1.4-fold) but not to UV (results not shown) suggesting a possibly unique sensitivity of FA-D1 fibroblasts to UV irradiation. This UV-sensitivity was still observed in SV40-immortalized FA-D1 fibroblasts and could be complemented by transfer of chromosome 13 providing BRCA2 (Fig. 2) suggesting that the observed UV-sensitivity is most probably due to the hypomorphic BRCA2 mutations in EUFA423 (Table 5). These results show that in general the cross-sensitivity of FA-D1/BRCA2 fibroblasts to various DNA damaging agents is very similar to that observed in fibroblasts from other FA complementation groups. The only exception was the significant sensitivity to UV, which may be unique for FA-D1/BRCA2 cells. 3.3. FA-D1/BRCA2 fibroblasts display RDS Inhibition of replicative DNA synthesis following ␥-radiation also known as the ionizing radiation (IR)inducible S phase checkpoint is observed in eukaryotic cells but not in cells derived from ataxia-telangiectasia and Nijmegen Breakage Syndrome patients [41]. A less pronounced inhibition of DNA synthesis is referred to as RDS. Since hamster cell mutants defective in Brca2 display RDS [38], we investigated this feature in FAD1/BRCA2 cells. FA-D1/BRCA2 primary fibroblasts appeared to display a significant RDS when compared to wild type fibroblasts (Fig. 3A), despite the lack of an increased IR-sensitivity in clonogenic survival in these cells (Fig. 1). A similar analysis in immortalized FA-D1 fibroblasts confirmed the presence of RDS, which was complemented by transfer of chromosome 13 providing wild type BRCA2 (Fig. 3B), indicating that BRCA2 is
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Fig. 1. FA-D1/BRCA2 fibroblasts display MMS, bleomycin and UV-sensitivity in addition to MMC-sensitivity. Clonogenic survival of wild type (VH10, FN1), FA-D1/BRCA2 (VU423, F145) and FA-D2 (EUFA8, EUFA1284, EUFA1289) primary fibroblasts following treatment with MMC, MMS, bleomycin and after irradiation with X-rays or UV. Data are the average of at least two independent experiments. Error bars represent the standard deviation (S.D.).
indeed involved in the above mentioned S-phase checkpoint. FA-A fibroblasts also showed the RDS phenotype albeit to a lesser extent than FA-D1 cells, whereas FAD2 fibroblasts showed near-normal inhibition of DNA synthesis in response to irradiation (Fig. 3A). Preliminary data with fibroblasts from a second FA-A patient
confirmed the observed RDS in the FA-A group (results not shown). This indicates that the IR-inducible S phase checkpoint is defective in FA-D1, FA-A and to a lesser extend in FA-D2 fibroblasts. RDS might therefore be a more general feature of FA-derived cells; additional complementation groups are currently being analyzed.
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Fig. 2. UV-sensitivity of FA-D1/BRCA2 cells can be complemented by transfer of chromosome 13 providing BRCA2. Clonogenic survival of SV40-immortalized wild type (VH10i), FA-D1/BRCA2 (EUFA423i) and FA-D1 fibroblasts after transfer of chromosome 13 (EUFA423i/#13) after irradiation with UV. Data are the average of at least three experiments; error bars represent the standard error of the mean (S.E.M.).
3.4. Spontaneous and MMC-induced CA FA patient-derived cells show increased chromosomal instability, which is further enhanced after treatment with MMC. This MMC-induced chromosome breakage
is used as a diagnostic test for the disease [1,2]. HRdeficient cells also display an increased frequency of spontaneous and MMC-induced CA [33–38,42]. Therefore, we studied CA in FA-D1/BRCA2 fibroblasts and B-cells to establish whether the degree of chromosomal
Fig. 3. FA-D1/BRCA2 fibroblasts display RDS in response to ␥-irradiation, which is complemented by transfer of chromosome 13 providing BRCA2. (A) Dose–response curve of the rate of DNA synthesis after ␥-irradiation in wild type (VH10, FN1), FA-A (EUFA432), FA-D1/BRCA2 (EUFA423, F145) and FA-D2 (EUFA1284, EUFA1289) primary fibroblasts. (B) Dose–response curve of the rate of DNA synthesis after ␥-irradiation in SV40-immortalized FA-D1/BRCA2 fibroblasts (EUFA432i) as well as in FA-D1 fibroblasts after transfer of chromosome 13 (EUFA423/#13). Data are the average of at least two experiments; error bars represent the S.E.M.
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Table 2 Relative sensitivities for spontaneous CA in primary fibroblasts
Relative sensitivity Spontaneous
VH10 (wt)
FN1 (wt)
EUFA432 (FA-A)
F145 (FA-D1)
EUFA423 (FA-D1)
1a
1.2
2.4b
8.9
5.6
The frequency of spontaneous CA per 100 cells in wild type VH10 cells was 19 ± 3, and this was set at 1. Both breaks and exchanges were counted as single aberrations. b For calculating relative sensitivities of mutant cell lines, the mean value of the two wild type lines was set at 1. a
instability of the FA-D1/BRCA2 cells resembles that of FA- or HR-deficient cells. FA-D1 as well as FA-A fibroblasts and BLCLs display higher levels of spontaneous CA than wild type cells (Tables 2, 3A and 3B). The majority of these aberrations in FA BLCL were of the chromatid type; breaks as well as exchanges were observed. In principle, two breaks are necessary to form an exchange configuration, thus exchanges used to be counted as two breaks when the total number of breaks was calculated. However, in the present study all dose–response relationships for MMC-induced exchange configurations turned out to be linear suggesting that single-hit events were underlying the exchange formation. Therefore, for calculating the total number of aberrations we gave equal weight to breaks and exchanges. When applying this method
to the spontaneous CA calculations this did not alter the outcome. Furthermore, the ratio of MMC-induced simple breaks versus exchanges varied from cell line to cell line, ranging from a value of 0.1 (TK6) to 1.5 (JVM, EUFA423). After MMC-treatment the relative chromosomal sensitivity of FA-A and FA-D1/BRCA2 BLCLs further increased to approximately 7-fold for HSC62 (FA-D1), 22.3-fold for HSC72 (FA-A) and to 56-fold for EUFA423 (FA-D1) BLCL (Table 3B) indicating that FA-D1 cells behave like FA-A derived cells. 3.5. Spontaneous and MMC-induced SCE formation Since, studies in HR-deficient rodent cells showed a reduced frequency of spontaneous and MMC-induced
Table 3A Spontaneous and MMC-induced CA per 100 B lymphoblastoid cells Cell line
Treatment
Cells with CA
Total breaksb ±S.E.M.
Chromatid Breaks
Exchangesa
JVM (wt)
Spontaneous 40 ng/ml MMC 320 ng/ml MMCc
2 7 28
2 4 17
0 3 23
2±1 10 ± 1 40 ± 7
TK6 (wt)
Spontaneous 40 ng/ml MMC 320 ng/ml MMC
1 21 53
2 23 102
0 3 15
2 ± 0.5 26 ± 3 117 ± 12
RAJI (wt)
Spontaneous 40 ng/ml MMC 320 ng/ml MMC
4 31 71
4 37 146
0 10 40
4 ± 0.5 47 ± 9 186 ± 1
HSC72 (FA-A)
Spontaneous 10 ng/ml MMC 20 ng/ml MMC
8 37 53
8 35 83
2 19 49
10 ± 5 54 ± 14 132 ± 19
HSC62 (FA-D1)
Spontaneous 10 ng/ml MMC 20 ng/ml MMC
6 23 34
4 19 38
2 18 21
6±3 37 ± 14 59 ± 12
EUFA423 (FA-D1)
Spontaneous 10 ng/ml MMC 20 ng/ml MMC
26 69 87
23 106 176
16 107 279
39 ± 3 213 ± 11 455 ± 41
a
Chromatid exchanges are interchanges, intrachanges, triradials or subchromatid exchanges. For calculations of the total number of breaks, exchanges were counted as 1 break. c The indicated frequencies of MMC-induced CA are after treatment with equitoxic doses of MMC giving similar inhibition of cell growth (0.005–1% survival). b
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Table 3B Relative sensitivities for CA in B lymphoblastoid cells
Relative sensitivity Spontaneous MMC-induced
JVM (wt)
TK6 (wt)
RAJI (wt)
HSC72 (FA-A)
HSC62 (FA-D1)
EUFA423 (FA-D1)
1a 1c
1 4.3
2 4.7
3.7b 22.3b
2.2 6.7
14.4 56
The frequency of spontaneous CA per 100 cells in wild type JVM B cells was 2 ± 1, and this was set at 1. For comparison with mutant cell lines the mean value of the three wild type cell lines was set at 1. All slopes were determined by using at least four different dose levels. c The slope of the induction curve of CA after MMC-treatment of wild type JVM B cells was 0.084 aberrations per 100 cells per ng/ml MMC, and this was set as 1. a
b
Fig. 4. FA-D1/BRCA2 lymphoblastoid cells show normal SCE induction after MMC treatment. Wild type (JVM, TK6, RAJI) and FA-A (HSC72) and FA-D1 (HSC62, EUFA423) patient derived (BLCL) were either mock-treated or treated with various MMC doses before SCE visualization. Data are the mean of at least two independent experiments; error bars represent the S.E.M.
SCE [33–35], we examined the frequency of spontaneous and MMC-induced SCE in FA-D1/BRCA2 B-cells. Levels of spontaneous SCE numbers in FA-D1 cells were not significantly different from wild type and FA-A B-cells (Fig. 4). After treatment with various doses of MMC, FA-D1/BRCA2 as well as FA-A B-cells showed a significant increase of SCE level up to 11–16 SCE/cell at a dose of 3.3 ng/ml (p < 0.001), which is in the same range as wild type cells showing 12–19 SCE/cell at a dose of 10 ng/ml (Fig. 4). This is also reflected by similar MMC-induced relative SCE responses, which are calculated from the slope of the dose–response curves, when comparing FA (2.1–2.8)
and wild type (0.8–1.8) cells (Table 4). These data indicate that FA-D1/BRCA2 B-cells are not hampered in the MMC-induced SCE formation like FA-A cells. 4. Discussion We document here the first detailed analysis of cells derived from two unrelated FA-D1/BRCA2 patients carrying biallelic mutations in BRCA2. Analysis of the CE and population doubling time revealed that the FA-D1 fibroblasts have CEs that are lower than those of other FA complementation groups, which is in agreement with other HR-deficient cells [25,33,36,42] but the observed
Table 4 Relative SCE responses
Relative sensitivity Spontaneous MMC-induced
JVM (wt)
TK6 (wt)
RAJI (wt)
HSC72 (FA-A)
HSC62 (FA-D1)
EUFA423 (FA-D1)
1a 1c
0.7 1.8
0.9 0.8
1.8b 2.1b
1.7 2.8
0.9 2.1
The frequency of spontaneous SCE per cell in wild type JVM B cells was 4.9 ± 0.3 and this was set at 1. For comparison with mutant cell lines the mean value of the three wild type cell lines was set at 1. All slopes were determined by using at least four different dose levels. c The slope of the induction curve of SCE after MMC-treatment in wild type JVM B cells was 0.85 SCE per ng/ml MMC, and this was set as 1. a
b
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Table 5 BRCA2 mutationsa in FA-D1 fibroblasts and/or B cell lines Cell lineb
Complementation group
Mutant allele 1 (exon)
Mutant allele 2 (exon)
F145 HSC62-L
FA-D1 FA-D1
IVS19-1 (20) G to A IVS19-1 (20) G to A
IVS19-1 (20) G to A IVS19-1 (20) G to A
EUFA423-F EUFA423-L
FA-D1 FA-D1
7691 insAT (15) 7691 insAT (15)
9900 insA (27) 9900 insA (27)
a b
BRCA2 mutations were previously described [7]. Cell lines F145 and HSC62-L are derived from the same FA patient; EUFA423-F and EUFA 423-L are also derived from the same patient.
longer population doubling time was also observed in other FA complementation groups. Our results revealed that the FA-D1/BRCA2 fibroblasts were slightly sensitive to MMS and bleomycin but not sensitive to X-ray irradiation, like cells derived from other FA groups. These data are in line with observations in FA cells from several complementation groups [43,44], but in contrast with data obtained in other BRCA2-deficient cells [38,42,45]. This discrepancy might be related to the hypomorphic BRCA2 mutations [7] found in these FA patients, which gives rise to truncated proteins that contain more functional domains when compared with other reported BRCA2-deficient cell lines. We observed some X-ray sensitivity in FAD2 line EUFA8, which might be caused by the genetic makeup of this patient since it was not observed in other unrelated FA-D2 patients. The sensitivity to bleomycin that was observed in all FA lines, suggests that bleomycin causes additional DNA lesions besides DNA double strand breaks (DSBs) that require the FA pathway for their repair. Moreover, we found that FA-D1 cells are markedly sensitive to UV irradiation and that this sensitivity could be complemented by transfer of chromosome 13 providing the BRCA2 gene, which is in concordance with other observations [38,42]. Taken together, these results indicate that in terms of cross-sensitivities FAD1/BRCA2-deficient cells display a FA-phenotype with unique UV-sensitivity. Notably, we observed that FA-D1/BRCA2 fibroblasts display RDS that also could be complemented by transfer of chromosome 13, which is in line with similar findings in Brca2-deficient hamster cells [38]. Surprisingly, we also observed RDS in FA-A and to a lesser extend in FA-D2 fibroblasts. These results suggest that in addition to ATM, MRE11, NBS1 and BRCA1 [41,46] also FANCD1/BRCA2 and possibly FANCA and FANCD2 [47] seem to be involved in the IR-inducible S-phase checkpoint. FA genes, including FANCD1/BRCA2, have also been implicated in the interstrand cross-link (ICL)inducible S-phase checkpoint, which depends on both ATR-CHK1 as well as ATR-NBS1-FANCD2 pathways
[48–50]. In a recent review, Bartek et al. [51] reported that at least three types of S-phase checkpoints exist. Nevertheless, it remains to be established if the ICLinduced S-phase checkpoint is also activated by DSBs like the IR-induced S-phase checkpoint or, alternatively, by stalled replication forks. FA cells and HR-deficient cells [33–38,42] display an increased frequency of spontaneous and MMC-induced CA, indicating that all these genes are involved in the maintenance of chromosomal stability, FANCD1/BRCA2 most probably via its direct function in HR [21–23]. The relative low level of MMC-induced CA in HSC62 (FA-D1) B-cells might be caused by the fact that the BRCA2 mutations (IVS19-1, exon 20; Table 5) in this patient are relatively mild resulting in a four amino acid shorter protein with most probably more residual function. SCE probably reflect post-replicational repair by HR that is associated with crossing-over between sister duplexes [52]. Thus, induction of SCE after MMC treatment was found to be impaired in HR-deficient cells including those that are BRCA2-deficient [33–38]. Surprisingly, we found that FA-D1/BRCA2 patient derived BLCL showed significant SCE induction in response to MMC, as was observed in wild type and FA-A cells most probably reflecting the residual BRCA2 function in these patients that seems to be sufficient to allow postreplicational repair to occur at a near-normal level. In the present paper we show that FA-D1/BRCA2 cells have features in common with cells from other FA complementation groups but also display some distinct characteristics: reduced CE and increased sensitivity to UV-irradiation that both could be complemented by transfer of the BRCA2 gene. These observations together with the previously reported impaired Rad51 foci formation in FA-D1 cells [30–32] separate the primary defective process in these cells from the general FA pathway and suggests this process to be more closely connected to the HR-pathway. This is in line with recently published data, which indicate that the FA pathway is separated from Rad51-mediated HR that
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requires BRCA2 [32]. Interestingly, also by their clinical phenotype FA-D1/BRCA2 patients are considered to represent a syndromic association distinct from typical FA, as they show a more severe disease progress with young-onset leukemia and solid tumors of the brain (medulloblastoma) and kidney (Wilms tumor) that are seldomly observed among FA patients from other complementation groups [53,54]. Acknowledgements We thank the FA families for participating; Drs. M. Buchwald and D. Papadopoulo for providing the F145 fibroblasts and the HSC62 lymphoblasts and Chantal van Paridon, Wouter Wiegant and Esther de Kruijf for their skilful technical assistance. This work was supported by a grant from the Dutch Cancer Foundation (KWF) (UL 2002–2739). References [1] A.D. Auerbach, M. Buchwald, H. Joenje, Fanconi anemia, in: B. Vogelstein, K.W. Kinzler (Eds.), The Genetic Basis of Human Cancer, McGraw-Hill, New York, 1998, pp. 317–332. [2] H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anemia, Nat. Rev. Genet. 2 (2001) 446–457. [3] M. Levitus, M.A. Rooimans, J. Steltenpool, N.F.C. Cool, A.B. Oostra, C.G. Mathew, M.E. Hoatlin, Q. Waisfisz, F. Arwert, J.P. de Winter, H. Joenje, Heterogeneity in Fanconi anemia: evidence for two genetic subtypes, Blood 103 (2004) 2498–2503. [4] C. Timmers, T. Taniguchi, J. Hejna, C. Reifsteck, L. Lucas, D. Bruun, M. Thayer, B. Cox, S. Olson, A. D’Andrea, Positional cloning of a novel Fanconi anemia gene, FANCD2, Mol. Cell 7 (2001) 241–248. [5] A.R. Meetei, J.P. de Winter, A.L. Medhurst, M. Wallisch, Q. Waisfisz, H.J. van de Vrugt, A.B. Oostra, Z. Yan, C. Ling, C.E. Bishop, M.E. Hoatlin, H. Joenje, W. Wang, A novel ubiquitin ligase is deficient in Fanconi anemia, Nat. Genet. 35 (2003) 165–170. [6] A.R. Meetei, M. Levitus, Y. Xue, A.L. Medhurst, M. Zwaan, C. Ling, M.A. Rooimans, P. Bier, M. Hoatlin, G. Pals, J.P. de Winter, W. Wang, H. Joenje, X-linked inheritance of Fanconi anemia complementation group B, Nat. Genet. 36 (2004) 1219–1224. [7] N.G. Howlett, T. Taniguchi, S. Olson, Q. Waisfisz, C. de DieSmulders, N. Persky, M. Grompe, H. Joenje, G. Pals, H. Ikeda, E.A. Fox, A.D. D’Andrea, Biallelic inactivation of BRCA2 in Fanconi anemia, Science 297 (2002) 606–609. [8] M. Levitus, Q. Waisfisz, B.C. Godthelp, Y. de Vries, S. Hussain, W.W. Wiegant, E. Elghalbzouri-Maghrani, J. Steltenpool, M.A. Rooimans, G. Pals, F. Arwert, C.G. Mathew, M.Z. Zdzienicka, K. Hiom, J.P. De Winter, H. Joenje, The DNA helicase BRIP1 is defective in Fanconi anemia complementation group, J. Nat. Genet. 37 (2005) 934–935. [9] A.R. Meetei, A.L. Medhurst, C. Ling, Y. Xue, T.R. Singh, P. Bier, J. Steltenpool, S. Stone, I. Dokal, C.G. Mathew, M. Hoatlin, H. Joenje, J.P. de Winter, W. Wang, A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M, Nat. Genet. 37 (2005) 958–963.
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Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2 Barbara C. Godthelp a , Paul P.W. van Buul a , Nicolaas G.J. Jaspers b , Elhaam Elghalbzouri-Maghrani a , Annemarie van Duijn-Goedhart a , Fr´e Arwert c , Hans Joenje c , Małgorzata Z. Zdzienicka a,d,∗ a
Department of Toxicogenetics, Leiden University Medical Center, Building 2, Postzone S-6-P, P.O. Box 9600, 2300 RC, Leiden, The Netherlands b Department of Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands c Department of Clinical Genetics and Human Genetics, Free University Medical Center, Amsterdam, The Netherlands d Department of Molecular Cell Genetics, Collegium Medicum, N.Copernicus University, Bydgoszcz, Poland Received 31 January 2006; received in revised form 4 July 2006; accepted 11 July 2006 Available online 21 August 2006
Abstract Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway. © 2006 Elsevier B.V. All rights reserved. Keywords: Fanconi anemia; BRCA2; Radioresistant DNA synthesis; DNA damaging agents; Chromosomal instability
1. Introduction FA is an autosomal recessively and X-linked inherited disorder characterized by multiple congenital abnormalities, abnormal skin pigmentation, progressive
∗
Corresponding author. Tel.: +31 71 5269614; fax: +31 71 5268284. E-mail address: [email protected] (M.Z. Zdzienicka).
0027-5107/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2006.07.003
bone marrow failure and a marked predisposition to develop cancer, primarily acute myeloid leukemia and squamous cell carcinomas of the head and neck. The cellular hallmark of FA is genomic instability and a specific hypersensitivity to DNA cross-linking agents such as MMC, which is used as a clinical diagnostic test for FA [1]. At least twelve different complementation groups can be discerned on the basis of complementation studies [2,3]. For 10 of these groups
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the disease-causing genes have been identified: FANCA, -B, -C, -D1/BRCA2, -D2, -E, -F, -G, -J and -L [2,4–8]. Recently, another FA gene was identified, FANCM, on basis of protein complex purification [9]. Despite the cloning of these FA genes, the precise function of the FA pathway is still largely unknown. The FA proteins are thought to function in a DNA damage response pathway involving the breast cancer susceptibility gene products, BRCA1 and BRCA2 [7,10]. A key step in this pathway is the activation of FANCD2 by monoubiquitination through FANCL [5] for which the nuclear multiprotein core complex consisting of FANCA, -B, -C, -E, -F, -G, -L and -M is required [2,5,6,9,11]. After monoubiquitination, FANCD2 is redistributed to nuclear foci containing BRCA1 and Rad51, linking FA with HR repair [10]. Moreover, the direct interaction of FANCA with BRCA1, of FANCG with FANCD1/BRCA2 and the interaction between ATM and FANCD2 [12–14], are also reminiscent of a role for the FA pathway in DNA repair, either directly or indirectly. Evidence for a more structural role of FA proteins comes from a model in which the function of FANCD2 monoubiquitination is to stabilize structurally blocked and broken replication forks prior to repair by specialized DNA damage repair pathways [15]. Since FA-D1/BRCA2 and FA-J patient derived cells display normal FANCD2 monoubiquitination following DNA damage, it is likely that these genes function independent of the FA core complex-FANCD2 pathway [3,7]. The recently identified genes, FANCJ and FANCM, both contain potential enzymatic domains belonging to the family of DEAH helicases or DNA stimulated ATPases and they are therefore likely to unwind and/or directly interact with DNA [8,9]. Germline mutations of the breast cancer susceptibility gene BRCA2 are associated with a predisposition to breast cancer but other cancers including ovarianand pancreatic-cancer are also seen in BRCA2 mutation carriers [16–18]. The BRCA2 protein is important in cellular responses to DNA damage and in maintaining genomic integrity, while a role in cell cycle progression has been suggested [19,20]. Crystal structure analysis revealed that BRCA2 is involved in Rad51-mediated HR as a stimulator that helps to displace RPA and facilitates ssDNA–Rad51 nucleoprotein filament formation [21,22]. Moreover, BRCA2 functions as a molecular switch since a decrease in BRCA2 phosphorylation stimulates its interaction with Rad51 [23]. Studying the role of BRCA2 in vivo has been proven difficult since knockout mice are embryonic lethals [24] but viable mice that harbor hypomorphic Brca2 mutations or conditional knockouts have been generated [25-27]. Until recently, CAPAN-1 derived from a pancreatic epithelial tumor
was the only well characterized human cell line defective in BRCA2 [28,29]. However, with the discovery that FAD1 patients carry biallelic mutations in BRCA2 [7], the first primary human BRCA2-deficient lines have become available, two of which are characterized in the present study (Table 5). These FA-D1/BRCA2 fibroblasts were found to be severely impaired in the formation of nuclear foci that contain the central HR protein, Rad51, a defect that has not been observed in any other FA complementation group [30–32]. Rodent and chicken cells that are deficient in HR genes, including BRCA2, display a severe genome instability phenotype with impaired DNA damage induced Rad51 foci formation, an excessive sensitivity to various DNA damaging agents, an increased frequency of spontaneous and MMC-induced chromatid-type chromosomal aberrations (CA) and a reduced capacity to form sister chromatid exchanges (SCE) [33–38]. To determine whether the phenotype of FAD1/BRCA2 cells conforms with that of HR repairdeficient cells or with that of “typical” FA cells, we analyzed the cellular characteristics of human BRCA2deficient fibroblasts and B-cells derived from two unrelated patients. As these cells displayed features that are considered characteristics of both FA- and HR-deficient cells, our results suggest that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls a separate pathway that is more directly linked to HR-based repair. 2. Materials and methods 2.1. Cell culture Primary fibroblasts were cultured in plastic dishes (P94 ; Greiner) in Ham’s F10 medium (GIBCO) without hypoxanthine and thymidine or in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (GIBCO), supplemented with 10% fetal calf serum (Bodinco), penicillin (100 U/ml) and streptomycin (0.1 mg/ml). Cells were maintained at 37 ◦ C in a 5% CO2 atmosphere, humidified to 95–100%. For subcultures the cells were detached by use of 0.25% trypsin containing 0.02% EDTA. The primary fibroblast strains used were: wild type (FN1, VH10), FA-A (EUFA432), FA-C (EUFA449), FA-D1 (EUFA423, F145), FA-D2 (EUFA8, EUFA1284, EUFA1289), FA-E (EUFA622), FA-F (EUFA121), AT (AT5BIVA) and the SV40-immortalized fibroblasts: wild type (MRC5i, VH10i), FA-D2 (PD733i) and FA-D1 (EUFA423i). B lymphoblastoid cell lines (BLCL): wild type (JVM, TK6, RAJI), FA-A (HSC72) and FA-D1 (HSC62, EUFA423-L) were grown in 75 cm2 culture flasks (Greiner) in RPMI 1640 medium Dutch modification (GIBCO) supplemented with GLUTAMAX I (GIBCO), 20 mM sodium pyruvate (GIBCO), 10% fetal calf serum (Bodinco), penicillin (100 U/ml) and streptomycin
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(0.1 mg/ml). BLCLs were maintained at 37 ◦ C in a 5% CO2 atmosphere, humidified to 95–100%. 2.2. Chemicals MMS was obtained from Merck, Darmstad, Germany; MMC from Kyowa, Hakko Kogyo, Tokyo; bleomycin from Dagra Pharma B.V.; polyethylene glycol (PEG, 1450 MW) and 5-bromo-2 -deoxyuridine (BrdUrd) were purchased from Sigma Chemical Co.
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sion was added to a monolayer of recipient cells (1.0 × 106 mutant cells per P94 dish) and allowed to attach at room temperature for 15 min. The cells were fused by treatment with 1 ml of 47% PEG in serum-free medium containing 10% DMSO for 1 min, followed by washing in serum-free medium containing 5% DMSO. After 24 h the cells were trypsinized and split to five P94 dishes with selective medium containing G418 (Gibco BRL). The resulting microcell hybrid clones were isolated after 14–28 days. 2.7. Analysis of CA, SCE
2.3. Irradiation For X-ray irradiation cells were irradiated in tissue culture medium at a dose rate of 2.98 Gy/min (200 kV, 4 mA, 1 mm Al). For gamma irradiation cells were irradiated with a 137 Cs source in culture medium at a dose rate of 0.8 Gy/min. For UVC irradiation (254 nm), a Philips TUV germicidal lamp was used with a fluence rate of 0.19 W/m2 , measured with the IL/770 germicidal radiometer; cells were irradiated in PBS. 2.4. Clonogenic survival assays Cultures in exponential growth were trypsinized and 500– 2000 fibroblasts were plated in P94 dishes in duplicate (controls in triplicate) left to attach for 4 h, and then irradiated or exposed to MMS for 1 h, bleomycin for 24 h and continuously to MMC, in complete medium. After treatment with chemicals the cells were washed twice with PBS, with the exception of MMC, and returned to fresh medium. After 14–17 days the dishes were rinsed with 0.9% NaCl, dried, stained with methylene blue (0.25%) and colonies were counted under a light microscope (LW Scientific). In all experiments, wild type fibroblasts were treated in an identical manner to serve as controls. 2.5. Measurement of radioresistant DNA synthesis (RDS) after ␥-irradiation Measurement of RDS was performed essentially as described but without the trichloroacetic acid precipitation step [39]. In short, cells were seeded in duplicate 30mm dishes (four for the unirradiated control) and were pre-labeled overnight with [14 C]-thymidine (Amersham) in HEPES-buffered thymidine-free Ham’s F10 medium and then exposed to graded doses of 137 Cs ␥-irradiation (1.2 Gy/min) and subsequently labeled with [3 H]-thymidine (Amersham) for 4 h. Free thymidine pools were chased by a further 30–45 min incubation in unlabeled medium. Scintillation counted 3 H/14 C radioactivity ratios of alkali-lysed cells were taken as a measure of DNA synthesis rates and plotted as percentages of unirradiated cells. 2.6. Microcell-mediated chromosome transfer Microcells with a single human chromosome 13 were obtained as described previously [38]. The microcell suspen-
Exponentially growing wild type and FA patient derived BLCL were either mock-treated or treated for 2 h (SCE) with MMC or for 24 h (CA) with 2, 3.3, 4, 5, 10, 16.5, 40, 80, 320 and 640 ng MMC/ml (wt) or with 1.0, 1.65, 2.0 and 3.3, 4, 10, 20 and 40 ng MMC/ml (FA cells). For SCE analysis, BrdUrd was added to the medium (10 M final concentration) to enable sister chromatid differentiation. The cells were harvested for CA analysis 24 h and for SCE analysis 51–72 h after (mock-) treatment, including 2 h incubation with colcemid (1 g/ml final concentration). The cells were fixed in ethanol:glacial acetic acid (4:1) after treatment with hypotonic solution (0.05 M KCl). Air-dried preparations were made and stained with a 5% aqueous Giemsa solution for 5 min to analyze CA and with FPG to visualize SCE [40]. For CA 100 mitotic cells were scored at each dose, while SCE were counted in 25 cells. Data for CA and SCE analyses are from at least two independent experiments of which the mean and the SEM were calculated. Statistical analysis was performed using t-tests.
3. Results 3.1. FA-D1/BRCA2 fibroblasts: population doubling times and cloning efficiencies We determined the cloning efficiency (CE) and population doubling time of FA-D1/BRCA2 primary fibroblasts to establish whether these FA-D1/BRCA2 cells resemble fibroblasts from other FA complementation groups. The data revealed that FA-D1/BRCA2 fibroblasts had a relatively low CE, i.e., 4.4 ± 0.9% (EUFA423) and 2.9 ± 0.98% (F145), respectively (Table 1), whereas all other FA fibroblasts showed CEs ranging from 8% in EUFA8 (FA-D2) to 26.7% in EUFA121 (FA-F), which is in range of wild type primary fibroblasts. In FA-D1 SV40-immortalized fibroblasts (EUFA423i) we also observed a lower CE that could be complemented to wild type level by transfer of a single human chromosome 13 providing the BRCA2 gene (Table 1). These FA-D1 cells with an additional chromosome 13 showed full complementation for MMC-sensitivity in cell survival and DNA damage induced nuclear Rad51 foci formation (results not shown). In addition, we
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Table 1 Low cloning efficiency in FA-D1/BRCA2 primary and immortal fibroblasts Cell line
Complementation group
Cloning efficiency (CE)a ±S.E.M.
VH10 VH25 FN1
wt wt wt
9.4 ± 1.7 18.9 ± 3.3 20.3 ± 3.4
MRC5ib VH10i
wti wti
38.5 ± 2.4 45.2 ± 17.6
EUFA432
FA-A
15.9 ± 2.1
EUFA449
FA-C
18.4 ± 2.5
EUFA423 F145
FA-D1/BRCA2 FA-D1/BRCA2
EUFA423i EUFA423i/#13
FA-D1/BRCA2i FA-D1 complemented
21.1 ± 1.6 34.7 ± 4.6
EUFA8 EUFA1284 EUFA1289
FA-D2 FA-D2 FA-D2
8.0 ± 0.53 22.0 ± 2.0 20.9 ± 1.8
PD733i
FA-D2i
34.0 ± 2.2
EUFA622
FA-E
23.5 ± 6.5
EUFA121
FA-F
26.7 ± 4.3
4.4 ± 0.90 2.9 ± 0.98
a The cloning deficiency was determined from at least six experiments. Results are the means ± S.E.M. b i, SV40-immortalized fibroblasts.
found that FA-D1/BRCA2 primary fibroblasts as well as FA-D2 fibroblasts have approximately a 2.3-fold longer population doubling time (≈135 h) than wild type fibroblasts (≈55 h), whereas e.g., FA-A fibroblasts exhibited an approximately 1.2-fold longer population doubling time (≈67 h). These results may suggest that primary cells carrying biallelic hypomorphic mutations in BRCA2 (Table 5) are more severely affected than cells that carry null mutations in any other FA gene. 3.2. Sensitivity to DNA damaging agents To determine cross-sensitivity of the FA-D1/BRCA2 fibroblasts to various DNA damaging agents, we compared the clonogenic survival of these cells in response to these agents with that of fibroblasts from other FA groups. FA-D1/BRCA2 as well as FA-D2 primary fibroblasts were approximately 5- (when compared to FN1) to 17-fold (when compared to VH10) more sensitive to MMC than wild type fibroblasts (Fig. 1), based on the dose required to reduce the survival to 10% (D10 ). Furthermore, we found no significant sensitivity to X-ray irradiation in fibroblasts from the FAD1/BRCA2 and FA-D2 groups (Fig. 1) when compared
with fibroblasts from an ataxia telangiectasia patient (AT5BIVA), which were ∼3-fold more sensitive than wild type fibroblasts. Similar observations were made for FA-A, FA-C and FA-J primary fibroblasts (results not shown). The only exception was EUFA8 (FA-D2) fibroblasts that showed a mildly increased sensitivity to X-ray irradiation (1.6-fold), which indicates that X-ray sensitivity in clonogenic survival is not a general feature of FA fibroblasts. Surprisingly, both FA-D1 and FA-D2 fibroblasts displayed a slightly increased sensitivity to the radiomimetic agent bleomycin (2-fold), which did not correspond with the lack of an increased X-ray sensitivity in clonogenic survival in these cells (Fig. 1). The FA-D1 and FA-D2 fibroblasts both showed a slightly increased sensitivity to the monofunctional alkylating agent MMS (∼1.4-fold), whereas only FA-D1 fibroblasts showed a marked sensitivity to UV irradiation (∼2-fold). Fibroblasts derived from FA-A, FA-E and FA-J complementation groups were also slightly sensitive to MMS (∼1.2–1.4-fold) but not to UV (results not shown) suggesting a possibly unique sensitivity of FA-D1 fibroblasts to UV irradiation. This UV-sensitivity was still observed in SV40-immortalized FA-D1 fibroblasts and could be complemented by transfer of chromosome 13 providing BRCA2 (Fig. 2) suggesting that the observed UV-sensitivity is most probably due to the hypomorphic BRCA2 mutations in EUFA423 (Table 5). These results show that in general the cross-sensitivity of FA-D1/BRCA2 fibroblasts to various DNA damaging agents is very similar to that observed in fibroblasts from other FA complementation groups. The only exception was the significant sensitivity to UV, which may be unique for FA-D1/BRCA2 cells. 3.3. FA-D1/BRCA2 fibroblasts display RDS Inhibition of replicative DNA synthesis following ␥-radiation also known as the ionizing radiation (IR)inducible S phase checkpoint is observed in eukaryotic cells but not in cells derived from ataxia-telangiectasia and Nijmegen Breakage Syndrome patients [41]. A less pronounced inhibition of DNA synthesis is referred to as RDS. Since hamster cell mutants defective in Brca2 display RDS [38], we investigated this feature in FAD1/BRCA2 cells. FA-D1/BRCA2 primary fibroblasts appeared to display a significant RDS when compared to wild type fibroblasts (Fig. 3A), despite the lack of an increased IR-sensitivity in clonogenic survival in these cells (Fig. 1). A similar analysis in immortalized FA-D1 fibroblasts confirmed the presence of RDS, which was complemented by transfer of chromosome 13 providing wild type BRCA2 (Fig. 3B), indicating that BRCA2 is
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Fig. 1. FA-D1/BRCA2 fibroblasts display MMS, bleomycin and UV-sensitivity in addition to MMC-sensitivity. Clonogenic survival of wild type (VH10, FN1), FA-D1/BRCA2 (VU423, F145) and FA-D2 (EUFA8, EUFA1284, EUFA1289) primary fibroblasts following treatment with MMC, MMS, bleomycin and after irradiation with X-rays or UV. Data are the average of at least two independent experiments. Error bars represent the standard deviation (S.D.).
indeed involved in the above mentioned S-phase checkpoint. FA-A fibroblasts also showed the RDS phenotype albeit to a lesser extent than FA-D1 cells, whereas FAD2 fibroblasts showed near-normal inhibition of DNA synthesis in response to irradiation (Fig. 3A). Preliminary data with fibroblasts from a second FA-A patient
confirmed the observed RDS in the FA-A group (results not shown). This indicates that the IR-inducible S phase checkpoint is defective in FA-D1, FA-A and to a lesser extend in FA-D2 fibroblasts. RDS might therefore be a more general feature of FA-derived cells; additional complementation groups are currently being analyzed.
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Fig. 2. UV-sensitivity of FA-D1/BRCA2 cells can be complemented by transfer of chromosome 13 providing BRCA2. Clonogenic survival of SV40-immortalized wild type (VH10i), FA-D1/BRCA2 (EUFA423i) and FA-D1 fibroblasts after transfer of chromosome 13 (EUFA423i/#13) after irradiation with UV. Data are the average of at least three experiments; error bars represent the standard error of the mean (S.E.M.).
3.4. Spontaneous and MMC-induced CA FA patient-derived cells show increased chromosomal instability, which is further enhanced after treatment with MMC. This MMC-induced chromosome breakage
is used as a diagnostic test for the disease [1,2]. HRdeficient cells also display an increased frequency of spontaneous and MMC-induced CA [33–38,42]. Therefore, we studied CA in FA-D1/BRCA2 fibroblasts and B-cells to establish whether the degree of chromosomal
Fig. 3. FA-D1/BRCA2 fibroblasts display RDS in response to ␥-irradiation, which is complemented by transfer of chromosome 13 providing BRCA2. (A) Dose–response curve of the rate of DNA synthesis after ␥-irradiation in wild type (VH10, FN1), FA-A (EUFA432), FA-D1/BRCA2 (EUFA423, F145) and FA-D2 (EUFA1284, EUFA1289) primary fibroblasts. (B) Dose–response curve of the rate of DNA synthesis after ␥-irradiation in SV40-immortalized FA-D1/BRCA2 fibroblasts (EUFA432i) as well as in FA-D1 fibroblasts after transfer of chromosome 13 (EUFA423/#13). Data are the average of at least two experiments; error bars represent the S.E.M.
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Table 2 Relative sensitivities for spontaneous CA in primary fibroblasts
Relative sensitivity Spontaneous
VH10 (wt)
FN1 (wt)
EUFA432 (FA-A)
F145 (FA-D1)
EUFA423 (FA-D1)
1a
1.2
2.4b
8.9
5.6
The frequency of spontaneous CA per 100 cells in wild type VH10 cells was 19 ± 3, and this was set at 1. Both breaks and exchanges were counted as single aberrations. b For calculating relative sensitivities of mutant cell lines, the mean value of the two wild type lines was set at 1. a
instability of the FA-D1/BRCA2 cells resembles that of FA- or HR-deficient cells. FA-D1 as well as FA-A fibroblasts and BLCLs display higher levels of spontaneous CA than wild type cells (Tables 2, 3A and 3B). The majority of these aberrations in FA BLCL were of the chromatid type; breaks as well as exchanges were observed. In principle, two breaks are necessary to form an exchange configuration, thus exchanges used to be counted as two breaks when the total number of breaks was calculated. However, in the present study all dose–response relationships for MMC-induced exchange configurations turned out to be linear suggesting that single-hit events were underlying the exchange formation. Therefore, for calculating the total number of aberrations we gave equal weight to breaks and exchanges. When applying this method
to the spontaneous CA calculations this did not alter the outcome. Furthermore, the ratio of MMC-induced simple breaks versus exchanges varied from cell line to cell line, ranging from a value of 0.1 (TK6) to 1.5 (JVM, EUFA423). After MMC-treatment the relative chromosomal sensitivity of FA-A and FA-D1/BRCA2 BLCLs further increased to approximately 7-fold for HSC62 (FA-D1), 22.3-fold for HSC72 (FA-A) and to 56-fold for EUFA423 (FA-D1) BLCL (Table 3B) indicating that FA-D1 cells behave like FA-A derived cells. 3.5. Spontaneous and MMC-induced SCE formation Since, studies in HR-deficient rodent cells showed a reduced frequency of spontaneous and MMC-induced
Table 3A Spontaneous and MMC-induced CA per 100 B lymphoblastoid cells Cell line
Treatment
Cells with CA
Total breaksb ±S.E.M.
Chromatid Breaks
Exchangesa
JVM (wt)
Spontaneous 40 ng/ml MMC 320 ng/ml MMCc
2 7 28
2 4 17
0 3 23
2±1 10 ± 1 40 ± 7
TK6 (wt)
Spontaneous 40 ng/ml MMC 320 ng/ml MMC
1 21 53
2 23 102
0 3 15
2 ± 0.5 26 ± 3 117 ± 12
RAJI (wt)
Spontaneous 40 ng/ml MMC 320 ng/ml MMC
4 31 71
4 37 146
0 10 40
4 ± 0.5 47 ± 9 186 ± 1
HSC72 (FA-A)
Spontaneous 10 ng/ml MMC 20 ng/ml MMC
8 37 53
8 35 83
2 19 49
10 ± 5 54 ± 14 132 ± 19
HSC62 (FA-D1)
Spontaneous 10 ng/ml MMC 20 ng/ml MMC
6 23 34
4 19 38
2 18 21
6±3 37 ± 14 59 ± 12
EUFA423 (FA-D1)
Spontaneous 10 ng/ml MMC 20 ng/ml MMC
26 69 87
23 106 176
16 107 279
39 ± 3 213 ± 11 455 ± 41
a
Chromatid exchanges are interchanges, intrachanges, triradials or subchromatid exchanges. For calculations of the total number of breaks, exchanges were counted as 1 break. c The indicated frequencies of MMC-induced CA are after treatment with equitoxic doses of MMC giving similar inhibition of cell growth (0.005–1% survival). b
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Table 3B Relative sensitivities for CA in B lymphoblastoid cells
Relative sensitivity Spontaneous MMC-induced
JVM (wt)
TK6 (wt)
RAJI (wt)
HSC72 (FA-A)
HSC62 (FA-D1)
EUFA423 (FA-D1)
1a 1c
1 4.3
2 4.7
3.7b 22.3b
2.2 6.7
14.4 56
The frequency of spontaneous CA per 100 cells in wild type JVM B cells was 2 ± 1, and this was set at 1. For comparison with mutant cell lines the mean value of the three wild type cell lines was set at 1. All slopes were determined by using at least four different dose levels. c The slope of the induction curve of CA after MMC-treatment of wild type JVM B cells was 0.084 aberrations per 100 cells per ng/ml MMC, and this was set as 1. a
b
Fig. 4. FA-D1/BRCA2 lymphoblastoid cells show normal SCE induction after MMC treatment. Wild type (JVM, TK6, RAJI) and FA-A (HSC72) and FA-D1 (HSC62, EUFA423) patient derived (BLCL) were either mock-treated or treated with various MMC doses before SCE visualization. Data are the mean of at least two independent experiments; error bars represent the S.E.M.
SCE [33–35], we examined the frequency of spontaneous and MMC-induced SCE in FA-D1/BRCA2 B-cells. Levels of spontaneous SCE numbers in FA-D1 cells were not significantly different from wild type and FA-A B-cells (Fig. 4). After treatment with various doses of MMC, FA-D1/BRCA2 as well as FA-A B-cells showed a significant increase of SCE level up to 11–16 SCE/cell at a dose of 3.3 ng/ml (p < 0.001), which is in the same range as wild type cells showing 12–19 SCE/cell at a dose of 10 ng/ml (Fig. 4). This is also reflected by similar MMC-induced relative SCE responses, which are calculated from the slope of the dose–response curves, when comparing FA (2.1–2.8)
and wild type (0.8–1.8) cells (Table 4). These data indicate that FA-D1/BRCA2 B-cells are not hampered in the MMC-induced SCE formation like FA-A cells. 4. Discussion We document here the first detailed analysis of cells derived from two unrelated FA-D1/BRCA2 patients carrying biallelic mutations in BRCA2. Analysis of the CE and population doubling time revealed that the FA-D1 fibroblasts have CEs that are lower than those of other FA complementation groups, which is in agreement with other HR-deficient cells [25,33,36,42] but the observed
Table 4 Relative SCE responses
Relative sensitivity Spontaneous MMC-induced
JVM (wt)
TK6 (wt)
RAJI (wt)
HSC72 (FA-A)
HSC62 (FA-D1)
EUFA423 (FA-D1)
1a 1c
0.7 1.8
0.9 0.8
1.8b 2.1b
1.7 2.8
0.9 2.1
The frequency of spontaneous SCE per cell in wild type JVM B cells was 4.9 ± 0.3 and this was set at 1. For comparison with mutant cell lines the mean value of the three wild type cell lines was set at 1. All slopes were determined by using at least four different dose levels. c The slope of the induction curve of SCE after MMC-treatment in wild type JVM B cells was 0.85 SCE per ng/ml MMC, and this was set as 1. a
b
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Table 5 BRCA2 mutationsa in FA-D1 fibroblasts and/or B cell lines Cell lineb
Complementation group
Mutant allele 1 (exon)
Mutant allele 2 (exon)
F145 HSC62-L
FA-D1 FA-D1
IVS19-1 (20) G to A IVS19-1 (20) G to A
IVS19-1 (20) G to A IVS19-1 (20) G to A
EUFA423-F EUFA423-L
FA-D1 FA-D1
7691 insAT (15) 7691 insAT (15)
9900 insA (27) 9900 insA (27)
a b
BRCA2 mutations were previously described [7]. Cell lines F145 and HSC62-L are derived from the same FA patient; EUFA423-F and EUFA 423-L are also derived from the same patient.
longer population doubling time was also observed in other FA complementation groups. Our results revealed that the FA-D1/BRCA2 fibroblasts were slightly sensitive to MMS and bleomycin but not sensitive to X-ray irradiation, like cells derived from other FA groups. These data are in line with observations in FA cells from several complementation groups [43,44], but in contrast with data obtained in other BRCA2-deficient cells [38,42,45]. This discrepancy might be related to the hypomorphic BRCA2 mutations [7] found in these FA patients, which gives rise to truncated proteins that contain more functional domains when compared with other reported BRCA2-deficient cell lines. We observed some X-ray sensitivity in FAD2 line EUFA8, which might be caused by the genetic makeup of this patient since it was not observed in other unrelated FA-D2 patients. The sensitivity to bleomycin that was observed in all FA lines, suggests that bleomycin causes additional DNA lesions besides DNA double strand breaks (DSBs) that require the FA pathway for their repair. Moreover, we found that FA-D1 cells are markedly sensitive to UV irradiation and that this sensitivity could be complemented by transfer of chromosome 13 providing the BRCA2 gene, which is in concordance with other observations [38,42]. Taken together, these results indicate that in terms of cross-sensitivities FAD1/BRCA2-deficient cells display a FA-phenotype with unique UV-sensitivity. Notably, we observed that FA-D1/BRCA2 fibroblasts display RDS that also could be complemented by transfer of chromosome 13, which is in line with similar findings in Brca2-deficient hamster cells [38]. Surprisingly, we also observed RDS in FA-A and to a lesser extend in FA-D2 fibroblasts. These results suggest that in addition to ATM, MRE11, NBS1 and BRCA1 [41,46] also FANCD1/BRCA2 and possibly FANCA and FANCD2 [47] seem to be involved in the IR-inducible S-phase checkpoint. FA genes, including FANCD1/BRCA2, have also been implicated in the interstrand cross-link (ICL)inducible S-phase checkpoint, which depends on both ATR-CHK1 as well as ATR-NBS1-FANCD2 pathways
[48–50]. In a recent review, Bartek et al. [51] reported that at least three types of S-phase checkpoints exist. Nevertheless, it remains to be established if the ICLinduced S-phase checkpoint is also activated by DSBs like the IR-induced S-phase checkpoint or, alternatively, by stalled replication forks. FA cells and HR-deficient cells [33–38,42] display an increased frequency of spontaneous and MMC-induced CA, indicating that all these genes are involved in the maintenance of chromosomal stability, FANCD1/BRCA2 most probably via its direct function in HR [21–23]. The relative low level of MMC-induced CA in HSC62 (FA-D1) B-cells might be caused by the fact that the BRCA2 mutations (IVS19-1, exon 20; Table 5) in this patient are relatively mild resulting in a four amino acid shorter protein with most probably more residual function. SCE probably reflect post-replicational repair by HR that is associated with crossing-over between sister duplexes [52]. Thus, induction of SCE after MMC treatment was found to be impaired in HR-deficient cells including those that are BRCA2-deficient [33–38]. Surprisingly, we found that FA-D1/BRCA2 patient derived BLCL showed significant SCE induction in response to MMC, as was observed in wild type and FA-A cells most probably reflecting the residual BRCA2 function in these patients that seems to be sufficient to allow postreplicational repair to occur at a near-normal level. In the present paper we show that FA-D1/BRCA2 cells have features in common with cells from other FA complementation groups but also display some distinct characteristics: reduced CE and increased sensitivity to UV-irradiation that both could be complemented by transfer of the BRCA2 gene. These observations together with the previously reported impaired Rad51 foci formation in FA-D1 cells [30–32] separate the primary defective process in these cells from the general FA pathway and suggests this process to be more closely connected to the HR-pathway. This is in line with recently published data, which indicate that the FA pathway is separated from Rad51-mediated HR that
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requires BRCA2 [32]. Interestingly, also by their clinical phenotype FA-D1/BRCA2 patients are considered to represent a syndromic association distinct from typical FA, as they show a more severe disease progress with young-onset leukemia and solid tumors of the brain (medulloblastoma) and kidney (Wilms tumor) that are seldomly observed among FA patients from other complementation groups [53,54]. Acknowledgements We thank the FA families for participating; Drs. M. Buchwald and D. Papadopoulo for providing the F145 fibroblasts and the HSC62 lymphoblasts and Chantal van Paridon, Wouter Wiegant and Esther de Kruijf for their skilful technical assistance. This work was supported by a grant from the Dutch Cancer Foundation (KWF) (UL 2002–2739). References [1] A.D. Auerbach, M. Buchwald, H. Joenje, Fanconi anemia, in: B. Vogelstein, K.W. Kinzler (Eds.), The Genetic Basis of Human Cancer, McGraw-Hill, New York, 1998, pp. 317–332. [2] H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anemia, Nat. Rev. Genet. 2 (2001) 446–457. [3] M. Levitus, M.A. Rooimans, J. Steltenpool, N.F.C. Cool, A.B. Oostra, C.G. Mathew, M.E. Hoatlin, Q. Waisfisz, F. Arwert, J.P. de Winter, H. Joenje, Heterogeneity in Fanconi anemia: evidence for two genetic subtypes, Blood 103 (2004) 2498–2503. [4] C. Timmers, T. Taniguchi, J. Hejna, C. Reifsteck, L. Lucas, D. Bruun, M. Thayer, B. Cox, S. Olson, A. D’Andrea, Positional cloning of a novel Fanconi anemia gene, FANCD2, Mol. Cell 7 (2001) 241–248. [5] A.R. Meetei, J.P. de Winter, A.L. Medhurst, M. Wallisch, Q. Waisfisz, H.J. van de Vrugt, A.B. Oostra, Z. Yan, C. Ling, C.E. Bishop, M.E. Hoatlin, H. Joenje, W. Wang, A novel ubiquitin ligase is deficient in Fanconi anemia, Nat. Genet. 35 (2003) 165–170. [6] A.R. Meetei, M. Levitus, Y. Xue, A.L. Medhurst, M. Zwaan, C. Ling, M.A. Rooimans, P. Bier, M. Hoatlin, G. Pals, J.P. de Winter, W. Wang, H. Joenje, X-linked inheritance of Fanconi anemia complementation group B, Nat. Genet. 36 (2004) 1219–1224. [7] N.G. Howlett, T. Taniguchi, S. Olson, Q. Waisfisz, C. de DieSmulders, N. Persky, M. Grompe, H. Joenje, G. Pals, H. Ikeda, E.A. Fox, A.D. D’Andrea, Biallelic inactivation of BRCA2 in Fanconi anemia, Science 297 (2002) 606–609. [8] M. Levitus, Q. Waisfisz, B.C. Godthelp, Y. de Vries, S. Hussain, W.W. Wiegant, E. Elghalbzouri-Maghrani, J. Steltenpool, M.A. Rooimans, G. Pals, F. Arwert, C.G. Mathew, M.Z. Zdzienicka, K. Hiom, J.P. De Winter, H. Joenje, The DNA helicase BRIP1 is defective in Fanconi anemia complementation group, J. Nat. Genet. 37 (2005) 934–935. [9] A.R. Meetei, A.L. Medhurst, C. Ling, Y. Xue, T.R. Singh, P. Bier, J. Steltenpool, S. Stone, I. Dokal, C.G. Mathew, M. Hoatlin, H. Joenje, J.P. de Winter, W. Wang, A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M, Nat. Genet. 37 (2005) 958–963.
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